Project Summary/Abstract Folding and assembly of proteins synthesized in the endoplasmic reticulum is closely monitored by a quality control apparatus that diverts folding-defective products to the cytosol to be degraded by the ubiquitin-proteasome system by a process known as endoplasmic reticulum-associated degradation (ERAD). The long-term goal of this project is to elucidate the mechanisms by which ERAD recognizes and destroys its targets. In the previous funding period we successfully implemented a large scale functional genomic analysis of the mammalian ERAD system that allowed us to perform unbiased analysis of substrate-selective ERAD in mammals. These data led to critical discoveries about the mechanisms of substrate triage and delivery to the HRD1 dislocon/ligase and the role of unconventional ubiquitin conjugation in coupling dislocation to degradation. The studies proposed in the present application harness state-of-the-art technologies that extend these discoveries and if successful will bring about a detailed molecular-level understanding of triage and quality control in the early secretory pathway. !